Publications by authors named "Barbara Mosca"

10 Publications

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Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER.

iScience 2021 Mar 3;24(3):102129. Epub 2021 Feb 3.

Department of Chemistry, Center for Diagnostics and Therapeutics, Advanced Translational Imaging Facility, Georgia State University, Atlanta, GA 30303, USA.

The precise spatiotemporal characteristics of subcellular calcium (Ca) transients are critical for the physiological processes. Here we report a green Ca sensor called "G-CatchER" using a protein design to report rapid local ER Ca dynamics with significantly improved folding properties. G-CatchER exhibits a superior Ca on rate to G-CEPIA1er and has a Ca-induced fluorescence lifetimes increase. G-CatchER also reports agonist/antagonist triggered Ca dynamics in several cell types including primary neurons that are orchestrated by IPRs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER-JP45), we report a rapid local Ca release that is likely due to calsequestrin. Transgenic expression of G-CatchER in muscle demonstrates its utility as an reporter of stimulus-evoked SR local Ca dynamics. G-CatchER will be an invaluable tool to examine local ER/SR Ca dynamics and facilitate drug development associated with ER dysfunction.
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http://dx.doi.org/10.1016/j.isci.2021.102129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900224PMC
March 2021

Role of the JP45-Calsequestrin Complex on Calcium Entry in Slow Twitch Skeletal Muscles.

J Biol Chem 2016 Jul 4;291(28):14555-65. Epub 2016 May 4.

From the Departments of Anaesthesia and Biomedicine, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland, Department of Life Science and Biotechnology, University of Ferrara, Via Borsari 46, 44100, Ferrara, Italy

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La(3+)- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca(2+) concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca(2+) concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.
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http://dx.doi.org/10.1074/jbc.M115.709071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938177PMC
July 2016

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies.

Hum Mol Genet 2015 Aug 27;24(16):4636-47. Epub 2015 May 27.

Department of Biomedicine and Anesthesia, Basel University Hospital, Basel, Switzerland, Department of Life Sciences, General Pathology Section, University of Ferrara, Ferrara, Italy,

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.
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http://dx.doi.org/10.1093/hmg/ddv195DOI Listing
August 2015

Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation-contraction coupling supramolecular complex.

Biochem J 2015 Feb;466(1):123-35

*Departments of Anesthesia and of Biomedicine, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland.

The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.
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http://dx.doi.org/10.1042/BJ20140935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843809PMC
February 2015

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice.

Nat Commun 2013 ;4:1541

Department of Experimental and Diagnostic Medicine, General Pathology section, University of Ferrara, Via Borsari 46, Ferrara 44121, Italy.

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.
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http://dx.doi.org/10.1038/ncomms2496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856057PMC
June 2013

Endogenously determined restriction of food intake overcomes excitation-contraction uncoupling in JP45KO mice with aging.

Exp Gerontol 2012 Apr 25;47(4):304-16. Epub 2012 Jan 25.

Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, United States.

The decline in muscular strength with age is disproportionate to the loss in total muscle mass that causes it. Knocking out JP45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), results in decreased expression of the voltage-gated Ca(2+) channel, Ca(v)1.1; excitation-contraction uncoupling (ECU); and loss of muscle force (Delbono et al., 2007). Here, we show that Ca(v)1.1 expression, charge movement, SR Ca(2+) release, in vitro contractile force, and sustained forced running remain stable in male JP45KO mice at 12 and 18 months. They also exhibit the level of ECU reported for 3-4-month mice (Delbono et al., 2007). No further decline at later ages was recorded. Preserved ECC was not related to increased expression of any protein that directly or indirectly interacts with JP45 at the triad junction. However, maintained muscle force and physical performance were associated with ablation of JP45 expression in the brain, spontaneous and significantly diminished food intake and less tendency toward obesity when exposed to a high-fat diet compared to WT. We propose that (1) endogenously generated restriction in food intake overcomes the deleterious effects of JP45 ablation on ECC and skeletal muscle force mainly through downregulation of neuropeptide-Y expression in the hypothalamic arcuate nucleus; and (2) the JP45KO mouse constitutes an invaluable model to examine the mechanisms controlling food intake as well as skeletal muscle function with aging.
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http://dx.doi.org/10.1016/j.exger.2012.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307889PMC
April 2012

SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase.

Biochem J 2012 Jan;441(2):731-41

Department of Anaesthesia, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland.

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²⁺ released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.
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http://dx.doi.org/10.1042/BJ20111457DOI Listing
January 2012

Remodeling of calcium handling in skeletal muscle through PGC-1α: impact on force, fatigability, and fiber type.

Am J Physiol Cell Physiol 2012 Jan 14;302(1):C88-99. Epub 2011 Sep 14.

Biozentrum, Department of Pharmacology/Neurobiology, University of Basel, Basel, Switzerland.

Regular endurance exercise remodels skeletal muscle, largely through the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α promotes fiber type switching and resistance to fatigue. Intracellular calcium levels might play a role in both adaptive phenomena, yet a role for PGC-1α in the adaptation of calcium handling in skeletal muscle remains unknown. Using mice with transgenic overexpression of PGC-1α, we now investigated the effect of PGC-1α on calcium handling in skeletal muscle. We demonstrate that PGC-1α induces a quantitative reduction in calcium release from the sarcoplasmic reticulum by diminishing the expression of calcium-releasing molecules. Concomitantly, maximal muscle force is reduced in vivo and ex vivo. In addition, PGC-1α overexpression delays calcium clearance from the myoplasm by interfering with multiple mechanisms involved in calcium removal, leading to higher myoplasmic calcium levels following contraction. During prolonged muscle activity, the delayed calcium clearance might facilitate force production in mice overexpressing PGC-1α. Our results reveal a novel role of PGC-1α in altering the contractile properties of skeletal muscle by modulating calcium handling. Importantly, our findings indicate PGC-1α to be both down- as well as upstream of calcium signaling in this tissue. Overall, our findings suggest that in the adaptation to chronic exercise, PGC-1α reduces maximal force, increases resistance to fatigue, and drives fiber type switching partly through remodeling of calcium transients, in addition to promoting slow-type myofibrillar protein expression and adequate energy supply.
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http://dx.doi.org/10.1152/ajpcell.00190.2011DOI Listing
January 2012

Minor sarcoplasmic reticulum membrane components that modulate excitation-contraction coupling in striated muscles.

J Physiol 2009 Jul 29;587(Pt 13):3071-9. Epub 2009 Apr 29.

Departments of Anesthesia and Biomedicine, Basel University Hospital, Switzerland.

In striated muscle, activation of contraction is initiated by membrane depolarisation caused by an action potential, which triggers the release of Ca(2+) stored in the sarcoplasmic reticulum by a process called excitation-contraction coupling. Excitation-contraction coupling occurs via a highly sophisticated supramolecular signalling complex at the junction between the sarcoplasmic reticulum and the transverse tubules. It is generally accepted that the core components of the excitation-contraction coupling machinery are the dihydropyridine receptors, ryanodine receptors and calsequestrin, which serve as voltage sensor, Ca(2+) release channel, and Ca(2+) storage protein, respectively. Nevertheless, a number of additional proteins have been shown to be essential both for the structural formation of the machinery involved in excitation-contraction coupling and for its fine tuning. In this review we discuss the functional role of minor sarcoplasmic reticulum protein components. The definition of their roles in excitation-contraction coupling is important in order to understand how mutations in genes involved in Ca(2+) signalling cause neuromuscular disorders.
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http://dx.doi.org/10.1113/jphysiol.2009.171876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727015PMC
July 2009